Method and kit for detection of anti-zika virus antibodies

ABSTRACT

Provided is a method of detecting the presence of an anti-Zika virus (ZIKV) antibody in a sample, including contacting a sample with a suspension having a plurality of microspheres wherein individual microspheres are conjugated to a peptide and the peptide includes a ZIKV peptide selected from the group including ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, forming a first incubated suspension by incubating said sample with said suspension to permit binding of anti-ZIKV antibodies present in the sample to said microspheres, forming a second incubated suspension by contacting said first incubated suspension with an anti-ZIKV antibody detecting-reagent to permit binding of the anti-ZIKV antibody detecting reagent to said microspheres, removing from the second incubated suspension anti-ZIKV antibody detecting-reagent molecules that are not bound to said microspheres, and detecting the presence of anti-ZIKV antibody detecting-reagent molecules in the second incubated suspension. Also provided is a kit containing reagents and compositions for performing the foregoing method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No. 62/421,694, filed Nov. 14, 2016, and this application claims priority to U.S. provisional application No. 62/438,681, filed Dec. 23, 2016, the contents of which applications are incorporated by reference in their entireties into the present disclosure.

FIELD OF THE INVENTION

The present disclosure relates to, inter alia, methods for detection of Zika virus infection and compositions of matter and kits for use in performing said method. More particularly, methods for detection of anti-Zika virus antibodies is provided. More particularly still, a method for detecting the presence of anti-Zika virus antibodies, and distinguished from the presence of antibodies to other flaviviruses. Also disclosed are kits for the performance of such methods.

BACKGROUND OF THE INVENTION

Zika virus (ZIKV) belongs to the genus of flavivirus within the family Flaviviridae. Many flaviviruses are significant human pathogens, including ZIKV, yellow fever (YFV), dengue virus (DENV serotypes 1 to 4), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). ZIKV is predominantly transmitted by Aedes spp. mosquitoes, which also transmit DENV and YFV, as well as chikungunya virus (an emerging alphavirus). Besides mosquitoes, ZIKV can also be transmitted through maternofetal route, sex, blood transfusion, and organ transplantation. Approximately 80% of the ZIKV infections are asymptomatic. Disease symptoms associated with ZIKV infection include headaches, fever, lethargy, rash, conjunctivitis, myalgia, and arthralgia. Severe diseases of ZIKV infection include neurotropic Guillain-Barre syndrome and congenital microcephaly. The flavivirus genome is a single-strand, positive-sense RNA of approximately 11,000 nucleotides. It contains a 5′ untranslated region (UTR), an open-reading frame (ORF), and a 3′ UTR. The single ORF encodes a long polyprotein which is processed into ten viral proteins, including three structural proteins—capsid (C), precursor membrane (prM), and envelope (E)—and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).

Diagnosis of ZIKV infection is performed through detection of viral components (e.g., viral RNA, viral proteins, or virus isolation) and detection of host immune response (e.g., antibodies against viral proteins). For viral component-based diagnosis, RT-PCR, immunoassay, and virus isolation detect ZIKV RNA, viral proteins, and live virus, respectively (Lanciotti et al., 2008); among them, RT-PCR is the most popular assay because of its sensitivity and specificity. The viremic phase of ZIKV infection usually lasts for about one week, yet occasionally persists beyond two weeks. Due to the short duration of the viremic phase, the diagnostic window for detection of viral components is narrow. Therefore, host immune response-based assays play an important role, among which enzyme-linked immunosorbent assays (ELISA), such as IgM-capture ELISA (MAC-ELISA), and plaque reduction neutralization test (PRNT) are the two most commonly used serologic assays in ZIKV diagnosis. Conventionally, serologic diagnosis of Zika virus (ZIKV) infection relies mainly upon IgM-capture ELISA which is confounded with the flaw of cross-reactivity among different flaviviruses. Unfortunately, the interpretation of conventional IgM-capture ELISA assays for ZIKV and other flaviviruses are challenging due to the cross-reactive nature of anti-flaviviral antibodies conventionally used in such tests, leading to equivocal diagnostic results. This challenge is confounding Zika diagnosis because (i) many flaviviruses (e.g., ZIKV and DENV) produce similar disease symptoms and (ii) antibodies from patients infected with ZIKV cross-react with other flaviviruses. Consequently, ZIKV IgM-capture ELISA results typically require neutralization tests for confirmation. Furthermore, PRNT is time-consuming, labor-intensive, slow, and low-throughput, and cost-ineffective, impairing attempts at rapid diagnosis to halt or slow spread if infection. Moreover, PRNT still relies upon both virus-specific and cross-reactive epitopes of viral E protein such that the results may be inconclusive with respect to flavivirus infections (Shan et al., 2016a). There is therefore a need to improve the accuracy and speed of serologic diagnosis for flaviviruses, ZIKV in particular.

SUMMARY OF THE INVENTION

The present disclosure relates to, inter alia, a method of detecting the presence of an anti-Zika virus (ZIKV) antibody in a sample, including contacting a sample with a suspension having a plurality of microspheres wherein individual microspheres are conjugated to a peptide and the peptide includes a ZIKV peptide selected from the group including ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, forming a first incubated suspension wherein forming includes incubating said sample with said suspension to permit binding of anti-ZIKV antibodies present in the sample to said microspheres, forming a second incubated suspension wherein forming includes contacting said first incubated suspension with an anti-ZIKV antibody detecting-reagent to permit binding of the anti-ZIKV antibody detecting reagent to said microspheres, removing from the second incubated suspension anti-ZIKV antibody detecting-reagent molecules that are not bound to said microspheres, and detecting the presence of anti-ZIKV antibody detecting-reagent molecules in the second incubated suspension.

In some embodiments, the method includes individual microspheres conjugated to ZIKV peptides that differ from ZIKV peptides to which other individual microspheres are conjugated, and the ZIKV peptides are selected from ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, and any two of the foregoing.

In other embodiments, different ZIKV peptides are ZIKV NS1 and ZIKV NS5. In some embodiments, individual microspheres are conjugated to ZIKV NS1, and in other embodiments individual microspheres are conjugated to ZIKV NS5. In some embodiments, some microspheres are conjugated to ZIKV Envelope protein, some microspheres are conjugated to ZIKV NS1, and some microspheres are conjugated to ZIKV NS5. In further embodiments, some microspheres are conjugated to ZIKV Envelope protein and some microspheres are conjugated to ZIKV NS1.

In other embodiments, the sample includes a biological sample from a subject and the biological sample is selected from the group including bodily fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissue homogenate, cellular extract, spinal fluid, and any combination of two or more of the foregoing. In other embodiments, the subject is a human.

In other embodiments, detecting includes measuring an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent, an autofluorescence signal emitted by a microsphere, or an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent and an autofluorescence signal emitted by a microsphere.

Some embodiments further include detecting the presence of an antibody to a Dengue virus (DENV) protein in a sample, wherein the protein comprises NS1 and the DENV comprises DENV1, DENV2, DENV3, DENV4, or any combination of two or more of the foregoing. For example, some microspheres may be conjugated to DENV1 NS1, some microspheres may be conjugated to DENV2 NS1, some microspheres may be conjugated to DENV3 NS1, and some microspheres may be conjugated to DENV4 NS1. In further embodiments, some microspheres may be conjugated to ZIKV Envelope protein, some microspheres may be conjugated to ZIKV NS1, some microspheres may be conjugated to ZIKV NS5, some microspheres may be conjugated to DENV1 NS1, some microspheres may be conjugated to DENV2 NS1, some microspheres may be conjugated to DENV3 NS1, and some microspheres may be conjugated to DENV4 NS1. In still further embodiments, some microspheres may be conjugated to ZIKV Envelope protein, some microspheres may be conjugated to ZIKV NS1, some microspheres may be conjugated to DENV1 NS1, some microspheres may be conjugated to DENV2 NS1, some microspheres may be conjugated to DENV3 NS1, and some microspheres may be conjugated to DENV4 NS1.

In another aspect, provided is a kit for detecting the presence of an anti-Zika virus (ZIKV) antibody in a sample, including a plurality of microspheres wherein individual microspheres are conjugated to a peptide and the peptide comprises a ZIKV peptide selected from the group consisting of ZIKV NS1, ZIKV NS5, and ZIKV envelope protein. In some embodiments, the kit includes individual microspheres conjugated to one of two or three different ZIKV peptides which differ from ZIKV peptides to which other microspheres are conjugated to, and the different ZIKV peptides are ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, or any two of the foregoing. In some embodiments, the different ZIKV peptides are ZIKV NS1 and ZIKV NS5. In other embodiments, the different ZIKV peptides are ZIKV envelope protein, ZIKV NS1, and ZIKV NS5. In further embodiments, the different ZIKV peptides are ZIKV envelope protein and ZIKV NS1. In still further embodiments, the kit may further include individual microspheres conjugated to one of two or more different DENV peptides which differ from DENV peptides to which other microspheres are conjugated to, and the different DENV peptides are DENV1 NS1, DENV2 NS1, DENV3, NS1, or DENV4 NS1. For example, a kit may include microspheres with the following peptides conjugated thereto: ZIKV NS1, ZIKV NS5, ZIKV Envelope protein, DENV1 NS1, DENV2 NS2, DENV3 NS3, and DENV4 NS4. In some examples, a kit may contain a subset of the foregoing. For example, a kit may contain microspheres with the following peptides conjugated thereto: ZIKV NS1, ZIKV Envelope protein, DENV1 NS1, DENV2 NS2, DENV3 NS3, and DENV4 NS1. In yet other embodiments, a kit may contain any combination of any two or more of the foregoing microspheres. For example, in some embodiments, the different ZIKV peptides are ZIKV NS1 and ZIKV NS5. In other embodiments, the different ZIKV peptides are ZIKV envelope protein, ZIKV NS1, and ZIKV NS5. In further embodiments, the different ZIKV peptides are ZIKV Envelope protein and ZIKV NS1.

In other embodiments, individual microspheres are conjugated to ZIKV NS1. In still other embodiments, individual microspheres are conjugated to ZIKV NS5. In still other embodiments, the kit includes an anti-ZIKV antibody-detecting reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating aspects of the present invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. Further, as provided, like reference numerals contained in the drawings are meant to identify similar or identical elements. The foregoing and other objects, features, and advantages of the invention are apparent from the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 shows SDS-PAGE analysis of recombinant ZIKV NS5 protein. Full-length NS5 of ZIKV was expressed in an E. coli system, and purified through affinity followed by size-exclusion chromatography (see details in Materials and Methods). The recombinant protein was analyzed on a 12% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-rad).

FIG. 2 shows a Table (Table 5) of a summary of PRNT, IgM-capture ELISA, and multiplex MIA diagnosis.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of an invention disclosed herein and certain features, advantages, and details thereof, are explained more fully below with reference to the non-limiting embodiments illustrated in the accompanying drawings. Descriptions of well-known materials, fabrication tools, processing techniques, etc., are omitted so as to not unnecessarily obscure the invention in detail. It should be understood, however, that the detailed description and the specific examples, while indicating some embodiments, are given by way of illustration only, and are not by way of limitation. Various substitutions, modifications, additions and/or arrangements within the spirit and/or scope of the underlying inventive concepts will be apparent to those skilled in the art from this disclosure.

Disclosed herein is a method for detecting the presence of anti-ZIKV antibodies in a sample, overcoming shortcomings of the prior art. Conventionally, detection of anti-ZIKV antibodies in a sample may require a high volume of sample, may take longer than several hours to perform, may be of low sensitivity resulting in a failure to detect a presence of anti-ZIKV antibodies in a sample, and low selectivity resulting in evidence of a presence of anti-ZIKV antibodies in a sample when no such antibodies are present. Such shortcomings may pose difficulties in accurate detection of the presence of anti-ZIKV antibodies in a sample. According to the present disclosure, small volumes of sample may be used to generate high-probability detection of anti ZIKV-antibodies in a sample.

In one aspect, disclosed is a method for detecting the presence of anti-Zika virus antibodies, such as in a sample from a subject. Such detection may indicate current or prior infection with ZIKV. In particular, the presence of antibodies to the NS5 protein of ZIKV may be detected. The presence of different antibody types, including IgG, IgM, or both, may be accomplished in accordance with the present disclosure. As would be understood by skilled artisans, the presence of different antibody types following a subject's exposure to ZIKV or other flaviviruses follows different time frames, with IgM antibodies to particular antigens being produced sooner after infection than IgG antibodies to said antigens. In various embodiments, detection of one, the other, both, and/or other types of antibodies to ZIKV-derived antigens may provide an indication of a time frame of a subject's exposure to ZIKV relative to when a sample was obtained from the subject. For example, presence of IgM anti-ZIKV antibodies may signify more recent exposure while presence of IgG anti-ZIKV antibodies may signify less recent exposure.

In another aspect, presence of antibodies to ZIKV NS5 may be performed. ZIKV NS5 protein is encoded for by nucleotides 7668-10376 of the ZIKV genome (GenBank number KU955593.1; Shan et al., 2016, Cell Host & Microbe 19:891-900). This sequence, and the peptide product thereof, ZIKV NS5 peptide, differ from the genetic and peptide sequences of NS5 for other viruses, including flaviviruses. For example, NS5 of DENV1-4 differ from ZIKV NS5. As disclosed herein, affinity of antibodies to ZIKV NS5 produced by a subject subsequent to exposure to ZIKV may differ from the affinity of said antibodies to NS5 protein of other viruses, such as DENV or other flaviviruses. The detection of the presence of an anti-ZIKV NS5 antibody in a subject on the basis of whether said antibody binds to ZIKV NS5 may therefore signify that the subject was exposed to ZIKV as distinguished from having been exposed to DENV. Specifically, identifying antibodies in a subject that bind ZIKV NS5 in accordance with the present disclosure indicates that the subject was exposed to ZIKV and may be distinguished from an indication that said subject was exposed to DENV, or other viruses such as other flaviviruses.

In another aspect, presence of antibodies to ZIKV NS1 may be detected. ZIKV NS1 protein is encoded for by nucleotides 2490-3545 of the ZIKV genome (see, e.g., GenBank accession number KU955593.1; Shan et al., 2016, Cell Host & Microbe 19:891-900). This sequence, and the peptide product thereof, ZIKV NS1 peptide, differ from the genetic and peptide sequences of NS1 for other viruses, including flaviviruses. For example, NS1 of DENV1-4 differ from ZIKV NS1. Gene and peptide sequence of NS1 for each of DENV1-4 also differ from each other. The complete genomes of DENV1, DENV2, DENV3, and DENV4 are known (see, e.g., GenBank accession numbers NC_001477.1, NC_001474.2, NC_001475.2, and NC_002640.1, respectively). As disclosed herein, affinity of antibodies to ZIKV NS1 produced by a subject subsequent to exposure to ZIKV differs from the affinity of said antibodies to NS1 protein of other viruses, such as DENV1-4 or other flaviviruses. Nucleotide sequences for DENV1-4 NS1, and the amino acid sequences of such proteins, is known (see, e.g., GenBank accession numbers NP_722461.1, NP_739584.2, YP_001531169.2, and NP_740318.1 for DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1, respectively).

In another aspect, presence of antibodies to a flavivirus envelope protein may be detected. ZIKV envelope peptide amino acid sequence is amino acids 291-792 of ZIKV polyprotein (GenBank accession number AAV34151). Envelope protein of other strains of ZIKV may also be used, as may Envelope proteins from other flaviviruses, such as DENV1 envelope protein, DENV2 envelope protein, DENV3 envelope protein, or DENV1 envelope protein, given the highly conserved amino acid sequences of envelope protein among these viruses and strains thereof.

As further disclosed herein, affinity of antibodies to DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1 produced by a subject subsequent to exposure to DENV1-4, respectively, may differ from the affinity of said antibodies to NS1 protein of other viruses, such as DENV serotypes to which the subject was not exposed, or other flaviviruses. The detection of the presence of an anti-ZIKV NS1 antibody in a subject on the basis of whether said antibody binds to ZIKV NS1 may therefore signify that the subject was exposed to ZIKV as distinguished from having been exposed to DENV1-4. Furthermore, the detection of the presence of an anti-DENV1 NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1 antibody in a subject on the basis of whether said antibody binds to DENV1 NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1, respectively, may therefore signify that the subject was exposed to DENV1, DENV2, DENV3, or DENV4, respectively, as distinguished from having been exposed to another serotype of DENV, ZIKV, or to another flavivirus. Specifically, as disclosed herein, identifying antibodies in a subject that bind ZIKV NS1 in accordance with the present disclosure indicates that the subject was exposed to ZIKV and may be distinguished from an indication that said subject was exposed to DENV, or other viruses such as other flaviviruses. And, as disclosed herein, identifying antibodies in a subject that bind to DENV1 NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1 in accordance with the present disclosure indicates that the subject was exposed to DENV1, DENV2, DENV3, or DENV4, respectively, and may be distinguished from an indication that said subject was exposed to another serotype of DENV, or to ZIKV or another flavivirus.

Antibodies produced in response to ZIKV exposure that have affinity for ZIKV Envelope protein (E) may also have affinity for E protein of other flaviviruses, such as DENV or other types of flaviviruses. Similarities of the peptide sequences and/or conformations of E protein of different flaviviruses may result in the production of antibodies that are cross-reactive to various flaviviruses. If a subject has been exposed to ZIKV, the subject may produce antibodies that have affinity not only for ZIKV E protein but also for E protein of other flaviviruses, including DENV. Likewise, a subject who has been exposed to DENV1-4 may produce antibodies with affinity for E protein of other flaviviruses. Detection in a subject of an antibody with affinity for ZIKV E protein may signify that the subject was exposed to ZIKV, DENV1, DENV2, DENV3, DENV4, or another flavivirus. And, detection in a subject of an antibody with affinity for DENV1 E, DENV2 E, DENV3 E, or DENV4 E may signify that the subject was exposed to ZIKV, DENV1, DENV2, DENV3, DENV4, or another flavivirus.

As further disclosed herein, in one embodiment, detection of multiple different antibodies may be performed on a given sample or samples from a given subject or subjects. In some embodiments, a sample from a subject may be tested for the presence of an antibody with affinity for ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, DENV4 NS1, or any combination of two or more of the foregoing. In one non-limiting example, a sample from a subject may be tested for the presence of antibodies to ZIKV NS5, ZIKV NS1, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1. In another non-limiting example, a sample from a subject may be tested for the presence of antibodies to ZIKV NS1, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1. The presence of IgG and IgM antibodies to the foregoing antigens may be tested. If a subject has been exposed to ZIKV but not to other flaviviruses, IgG antibodies, IgM antibodies, or both , to ZIKV NS1, ZIKV NS5, and ZIKV E may be detected. If the subject has been exposed to DENV1-4, antibodies to DENV1-4 NS1 may be detected, as well as to ZIKV E owing to the similarities between ZIKV E and DENV E and cross-reactivity of antibodies thereto. If a subject has been exposed to both ZIKV and DENV, antibodies to ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1 may all be detected in a sample or samples from said subject.

In a particular embodiment, presence or absence of anti-ZIKV NS5 antibodies may be detected in a sample from a subject. As disclosed herein, high selectivity of anti-ZIKV NS5 antibodies for ZIKV NS5 relative to NS5 or other proteins from other flaviviruses, such as DENV, corresponds to low cross-reactivity of anti-ZIKV NS5 antibodies for other flaviviruses. Similarly, as disclosed herein, low cross-reactivity of antibodies produced in response to exposure to flaviviruses other than ZIKV, such as DENV, to ZIKV NS5 results from selectivity of antibodies produced in response to expose to said other viruses relative to ZIKV NS5. Detection of the presence of an anti-ZIKV NS5 antibody in a sample from a subject as disclosed herein indicates that the subject has been exposed to ZIKV. The absence of anti-ZIKV NS5 antibodies in a sample from a subject may signify that the subject was not exposed to NS5, even if the subject was exposed to another virus, such as another flavivirus such as DENV.

In another particular embodiment, presence or absence of anti-ZIKV NS1 antibodies may be detected in a sample from a subject. As disclosed herein, high selectivity of anti-ZIKV NS1 antibodies for ZIKV NS1 relative to NS1 or other proteins from other flaviviruses, such as DENV, corresponds to low cross-reactivity of anti-ZIKV NS1 antibodies for other flaviviruses. Similarly, as disclosed herein, low cross-reactivity of antibodies produced in response to exposure to flaviviruses other than ZIKV, such as DENV, to ZIKV NS1 results from selectivity of antibodies produced in response to expose to said other viruses relative to ZIKV NS1. Detection of the presence of an anti-ZIKV NS1 antibody in a sample from a subject as disclosed herein indicates that the subject has been exposed to ZIKV. The absence of anti-ZIKV NS1 antibodies in a sample from a subject may signify that the subject was not exposed to NS1, even if the subject was exposed to another virus, such as another flavivirus such as DENV.

In another particular embodiment, presence or absence of anti-ZIKV envelope protein antibodies may be detected in a sample from a subject. In particular examples, presence or absence of anti-ZIKV NS1, anti-ZIKV NS5, anti-ZIKV envelope protein, anti-DENV1 NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 may all be detected in a sample from a subject. In another example, presence or absence of anti-ZIKV NS1, anti-ZIKV envelope protein, anti-DENV1 NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 may all be detected in a sample from a subject.

In some embodiments, for each and every of the anti-flaviviral protein antibodies disclosed herein, an assay for detection thereof may generate some level of signal indicating presence of reactive antibody in a sample from a subject known not to have been exposed to a or the flaviviruses of interest. Such background signal is known by those skilled in this field to occur and is attributable to some baseline reactivity of sample constituents with a test antigen used as disclosed herein for identifying presence of an anti-flaviviral antibody. Conventionally, an average level of such background may be determined for a given assay against which detection results for a tested subject may be compared to determine whether a given antibody is present. For example, a number of subjects known or believed not to have been exposed to ZIKV or DENV may be tested for the presence of anti-ZIKV and anti-DENV protein antibodies as disclosed herein and an average level of background reactivity of assays as disclosed herein may be determined for a given assay.

For subsequent testing of a subject who may or may not express anti-ZIKV or anti-DENV protein antibodies, a sample taken from such subject may be tested in an assay as disclosed herein and results compared to an average from tests of subjects known or believed not to have been exposed to such flaviviruses. In some instances, a level of detection signal some degree or amount above an average background level of expression may be selected as minimum level qualifying for evincing presence of a given anti-flaviviral protein antibody in a sample from a subject. If a subject is tested for the presence of antibodies against one or more of the foregoing flaviviral proteins and the level of detection signifying presence of any one or more of such antibodies falls below, or above, such cutoff value, then such antibodies may be determined not to have been detected, or to have been detected, respectively, in such sample. In some examples, a cutoff value may be a certain degree above an average level of background signal detected in subjects known or believed not to have been exposed to the flaviviruses in question. For example, a cutoff may be a number of standard deviations above such mean background. A cutoff value may be one, two, or three standard deviations above mean background expression, meaning that antibody to a given flaviviral protein is determined to have been detected for a sample when an assay yields a given level of signal signifying such presence. Cutoff levels may be determined on an anti-flaviviral protein antibody by anti-flaviviral protein antibody basis, as well as on an assay-by-assay basis.

In some embodiments, if a test detects presence of anti-flaviviral proteins, a test may be repeated at a later date and comparing indications that such antibodies were detected at different tests performed. For example, presence or absence of an IgG antibody, or an IgM antibody, or both types of antibodies, to one or more of the flaviviral proteins disclosed herein, may be detected. For example, IgM antibodies to ZIKV envelope and ZIKV NS1, may be detected. Such results may indicate current or recent infection with ZIKV, such as in approximately the past 21 days, as would detection of IgM antibodies to ZIKV NS5. In another example, IgG antibodies to ZIKV envelope and ZIKV NS1, may be detected. Such results may indicate past infection with ZIKV, such as more than 21 days ago, as would detection of IgG antibodies to ZIKV NS5. In still another example, IgM and IgG antibodies to ZIKV envelope and ZIKV NS1, may be detected. Such results may indicate current or recent infection with ZIKV, such as in approximately the past 21 days, as well as past infection, such as more than approximately 21 days ago, as would detection of IgM and IgG antibodies to ZIKV NS5.

In still further embodiments, an avidity assay may be performed to determine whether an anti-flaviviral protein antibody, such as anti-ZIKV envelope protein, anti-ZIKV-NS1, anti-ZIKV NS5, anti-DENV1 NS1, anti-DENV2 NS2, anti-DENV3 NS1, or anti-DENV4 NS1 antibody, or any combination of two or more of the foregoing antibodies, are present and if so, whether they bind to ZIKV envelope protein, ZIKV-NS1, ZIKV NS5, DENV1 NS1, DENV2 NS2, DENV3 NS1, or DENV4 NS1, respectively, with high or low avidity. Detection of high-avidity-binding antibodies that bind to a flaviviral protein with relatively higher avidity (e.g., anti-ZIKV envelope/ZIKV envelope, anti-ZIKV NS1/ZIKV/NS1, anti-ZIKV NS5/ZIKV NS5, anti-DENV1 NS1/DENV1 NS1, anti-DENV2 NS1/DENV2 NSI, anti-DENV3 NS1/DENV3 NS1, or anti-DENV4 NS1/DENV4 binding) may indicate a subject was infected with a given flavivirus as disclosed herein longer ago. Detection of low-avidity-binding antibodies that bind to a flaviviral protein with relatively low avidity (e.g., anti-ZIKV envelope/ZIKV envelope, anti-ZIKV NS1/ZIKV/NS1, anti-ZIKV NS5/ZIKV NS5, anti-DENV1 NS1/DENV1 NS1, anti-DENV2 NS1/DENV2 NSI, anti-DENV3 NS1/DENV3 NS1, or anti-DENV4 NS1/DENV4 binding) may indicate a subject was infected with a given flavivirus more recently, or is currently infected with a given flavivirus. High avidity binding is binding that persists or is still detectable after incubation of a solution of a flaviviral protein or composition such as a polyplex microsphere bound to such antigen as disclosed herein with urea. Urea is known to disrupt low-avidity antigen-antibody association. When a sample is incubated with urea (e.g., 6 M, 8 M, or 10 M urea for 10 minutes) yet presence of anti-flaviviral antibody as disclosed herein is still detected, then high-avidity binding antibodies that bind to a given flaviviral protein have been detected. Detection of absence of antibody after urea incubation indicates absence of high-avidity antibodies. Such avidity testing may be performed for any one, all or any combination of assays for presence of anti-flaviviral protein antibodies.

In some embodiments, presence or absence of a given anti-flaviviral protein antibody or antibodies may indicate that a subject from which a tested sample was taken was previously exposed to or infected with or whose body presently or formerly contained a given flavivirus. For example, if anti-ZIKV envelope protein antibodies are detected in a sample, a subject may be determined to have had or presently have a flavivirus. Given cross-reactivity of an anti-ZIKV envelope protein antibody with envelope proteins of other flaviviruses, such as DENV1, DENV2, DENV3, or DENV4, for example, apparent detection of an anti-ZIKV envelope antibody may signify that the subject has had or has ZIKV, DENV1, DENV2, DENV3, or DENV4. By contrast, as disclosed herein, given selectivity of antibodies to ZIKV NS1 to binding to ZIKV NS1 relative to binding to other flaviviral proteins, such as DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1, which is low, detection of anti-ZIKV NS1 antibodies as disclosed herein signifies that a subject has or has had ZIKV in particular. Similarly, as disclosed herein, given selectivity of antibodies to ZIKV NS5 to binding to ZIKV NS5 relative to binding to other flaviviral proteins, such as DENV1 NS5, DENV2 NS5, DENV3 NS5, or DENV4 NS5, which is low, detection of anti-ZIKV NS5 antibodies as disclosed herein signifies that a subject has or has had ZIKV.

As further disclosed herein, detection of presence multiple different anti-flaviviral protein antibodies combined with detection of absence of e multiple different anti-flaviviral protein antibodies in combination permits possible discriminative determination of presence, or prior presence, of ZIKV, DENV, or both, in a subject. For example, a subject may have had or may have ZIKV but not DENV of any serotype, in which case presence of anti-ZIKV NS1, anti-ZIKV-NS5, and anti-ZIKV envelope protein antibodies may be determined to be present in a sample from such subject, whereas anti-DENV NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 antibodies may be determined not to be present in samples from such patients. In another example, a subject may have had or may have a flavivirus of a DENV serotype but not ZIKV, in which case absence of anti-ZIKV NS1 and anti-ZIKV-NS5 antibodies may be detected, and presence of anti-ZIKV envelope protein antibodies, and anti-DENV NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 antibodies (depending on which DENV serotype the subject had or has), may be determined to be present in samples from such patients.

The presence of an antibody to a given antigen as disclosed herein may be performed by any of a number of different known methods. In one embodiment, a microsphere immunoassay (MIA) may be used for the detection of an antibody to a given antigen. As would be appreciated by skilled artisans, rapid detection of multiple assays can be performed on low volumes of specimen taken from a subject. In some embodiments, presence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4 may be identified in a sample from a subject. In accordance with above-identified differential responsiveness to such testing on the basis of prior viral exposure, different combinations of positive results for particular antibodies may indicate exposure of the subject to ZIKV, DENV1-4, or another flavivirus.

Several options for MIA are available. In some embodiments, flaviviral antigens may be covalently coupled to microbeads and contacted to a sample from a subject, whereupon antibodies to the given antigen present in the sample bind to the bead via the antigen. Subsequent identification of antibody bound to the bead indicates that antibodies to the antigen were present in the sample. In other embodiments, antigen may be coupled to microbeads that have an anti-antigen antibody, or other molecule designed to bind to the antigen such as an affimer or aptamer or other designer ligand, fused thereto via binding to said antibody. Subsequent contacting of the antigen-coupled microbeads to sample from a subject may then be performed as described.

Different sources of viral antigen may be used in accordance with the present disclosure. For example, recombinant peptides whose sequence correspond to the sequence of viral peptide of interest (i.e., ZIKV NS1, ZIKV NS5, ZIKV E, or NS1 proteins of any of DENV1-4) may be synthesized by well-known methods and purified. Recombinant peptides may differ in sequence from native of wild-type flaviviral protein, provided it retains structural, sequential, or conformational properties sufficient to permit recognition by antibodies generated in a subject subsequent to exposure to the corresponding flavivirus. flaviviral protein from which the to which the recombinant Different assays may detect whether antibodies to flaviviral antigen in a sample are IgG antibodies or IgM antibodies, by known methods (e.g., IgG- or IgM-specific methods of detection of anti-flaviviral antigen antibody bound to microbeads, depleting IgG or IgM antibodies from a sample before contacting the sample with antigen-coupled beads, or selecting out IgG or IgM antibodies from a sample and contacting the selected-out antibodies with antigen-coupled microbeads.

In another embodiment, ELISA could be used to detect the presence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4 in a sample. For example, anti-IgM antibodies can be coated onto a plate, such as a 96-well or other multi-well plate, and contacted with sample from a subject. IgM antibodies from the sample would become bound to the anti-IgM-coated plate. Following well-known protocols, the IgM-bound plate could then be contacted with antiviral protein of interest, such as ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4, followed by contact with a visualizable antibody to the viral protein. In this way, antiviral protein of interest would, via binding to any antibodies thereto present in the sample that had become bound to the anti-IgM antibody on the plate, be bound to plates that had been exposed to samples from subject that had been exposed to the flavivirus. In turn, such samples would be visualizable, via binding of visualizable anti-flaviviral protein, such as through conjugation with antibodies, fluorophores, or other well-known visualizable or detectable markers. Upon processing to detect presence of the visualizable marker, plates that had been contacted with a sample from a subject that had been exposed to the corresponding flavivirus can be identified and distinguished from those that have not. By performing MAC-ELISA on a sample for antibodies to several different flaviviral proteins (for example, any combination of two or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4), it can be determined whether the subject had been exposed to ZIKV, DENV, and/or another flavivirus.

In another embodiment, a dot blot may be performed to detect the presence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4. In dot blotting, a spot or dot of antigen is applied to a membrane, such as nitrocellulose or polyvinyidine fluoride (PVDF). The membrane may then be exposed to a sample from a subject. If the subject has been exposed to a virus that contains the antigen applied to the membrane, such antibodies will couple to a region of the membrane to which the antigen has been applied. The membrane can then be contacted by secondary antibodies (e.g., anti-IgM or anti-IgG) coupled to a visualizable marker (e.g., radiolabel, fluorescent tag, magnetic tag, enzyme, etc.). Visualizable marker is thereby coupled to any region of the membrane to which an antigen recognized by antibodies thereto present in the sample. Upon detection of said marker, presence of antibodies to said antigen in the sample may be determined. In some embodiments, a dot blot analysis may be performed to detect the presence of antibodies to any one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4, and any combination of two or more of the foregoing. As described above, different permutations of positive and negative results for different anti-flaviviral antigen antibodies suggests that the subject from which the sample was taken was exposed to different flavivirus(es).

In yet another embodiment, immunochromatography may be used to identify the presence of one or more flaviviral antibody in a subject. As one nonlimiting example, and immunochromatographic strip test (ICST) may be used. In such assays, a small strip comprising capillary beds may be exposed to a sample from a subject. Embedded in the strip may be a viral antigen to which antibodies are to be detected. When an aqueous sample is drawn into contact with such antigens following contact with the capillary structure of the strip, the antigens are bound to any antibodies thereto that are present in the sample. The antigen may carry visualizable markers, such as colored latex, gold nanoparticles, or fluorescent or magnetic particles which can be visualized with appropriate readers. Further along the strip, a capture portion containing antibodies to the antigen immobilized to the strip bed may be present, such as in a concentrated region, stripe, or area. As sample continues to migrate along the capillary bed, it encounters the immobilized antibodies of the capture portion. If antibody to the particular antigen were present in the sample, their migration across the capillary bed would have carried bound, visualizable antigen with them, which, in turn, would be immobilized on the capture portion owing to the anti-antigen antibodies immobilized therein. As more antibody accumulates that the capture portion, the visualizable markers become detectable and, when detected, signify the presence of antibody to the antigen of interest.

In some examples, multiple ICST strips, each designed to permit the detection of antibodies to a different flaviviral antigen (e.g., ZIKV NS1, ZIKV NS5, ZIKV E, or NS1 proteins of any of DENV1-4) may be used to test the presence of antibodies to more than one such antigen in a sample. As described above, different permutations of positive and negative results for different anti-flaviviral antigen antibodies suggests that the subject from which the sample was taken was exposed to different flavivirus(es).

As would be appreciated by skilled artisans, modifications of the foregoing methods may be performed in keeping the with present disclosure. Other methods where ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, DENV4 NS1, or combinations of any two or more of the foregoing, are used to bind antibodies present in a sample, followed by methods used to visualize, detect, quantify, bind, identify, or otherwise record the presence and/or quantity thereof in the sample may be performed, all of which are explicitly intended as embodiments of the present disclosure, of which the foregoing examples are but non-limiting representations of the method disclosed herein. Kits containing reagents used in performing any such method, including reagents, antigens, labeling reagents or labeled antigens or antibodies, microspheres, binding membranes such as nitrocellulose, PVDF, or the like, lateral flow strips, with or without reagents needed for performing the disclosed method included therein, any tubes, vials, needles, or other tools needed for obtaining and/or storing samples, etc., are explicitly included in and considered embodiments of an invention disclosed herein.

As would be understood by skilled artisans, antigens used in any of the foregoing methods may be modified in accordance with standard molecular biology techniques. For example, antigens may be produced with identifiable tags appended thereto to facilitate isolation, identification, concentration, collection, application, or other uses thereof. As one non-limiting example, antigens with histidine tags may be produced to facilitate separation of recombinant antigens from other constituents employed during synthesis thereof, and subsequent concentration of said antigens in a usable form, solvent, diluent, or concentration. Although histidine tags are mentioned here as one non-limiting example, skilled artisans would appreciate that any of a wide variety of such tools could be adapted to and employed with the above-disclosed methods and compositions, without departing from, and hereby explicitly recounted as included within, the method as disclosed herein.

Although non-limiting examples discussed herein include using full-length recombinant flaviviral peptides as antigens, skilled artisans would appreciate that full-length peptides would not necessarily be required for use in all embodiments of the method and compositions disclosed herein. For example, a specific or portions less than full-length viral proteins may be used rather than full-length protein. A portion less than full-length protein to which antibodies produced in response to a subject's exposure to a given flavivirus will bind may be used rather than full-length protein, for example. In some embodiments, a portion less than full-length flaviviral protein to which antibodies produced in response to a subject's exposure to a given flavivirus, but not another flavivirus, will bind may be used. In other examples, portions less than full length protein that bind to antibodies produced in response to either of two or more different flaviviruses may be used.

In yet other embodiments, an antigen with less than total homology to a flaviviral protein, such as ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1 may be used, as may a combination of any two or more of the foregoing. For example, a recombinant flaviviral protein with an amino acid sequence that corresponds to some but not all of the amino acids in the flaviviral protein to which it corresponds may be used. In some examples, a recombinant flaviviral antigen with 75% or more homology to a corresponding naturally occurring flaviviral protein may be used. In some examples, a flaviviral antigen with between 75%-80% homology, 80%-85% homology, 85%-90% homology, 90%-95% homology, 95%-100% homology, 90%-91% homology, 91%-92% homology, 92%-93% homology, 93%-94% homology, 94%-95% homology, 95%-96% homology, 96%-97% homology, 97%-98% homology, or 99%-100% homology may be used. Several strains of ZIKV have been identified with high levels of homology between their sequences of ZIKV envelope protein, between their sequences of ZIKV NS1, and between their sequences of ZIKV NS5. ZIKV protein sequences homologous to such known strains may be used, within homology ranges as disclosed herein, in accordance with the present disclosure. A recombinant flaviviral antigen with less than 100% homology to a corresponding flaviviral protein to which antibodies produced in response to a subject's exposure to a given flavivirus will bind may be used rather than a recombinant protein with 100% homology. In some embodiments, a recombinant flaviviral protein of less than 100% homology to flaviviral protein to which antibodies produced in response to a subject's exposure to a given flavivirus, but not another flavivirus, will bind may be used. In other examples, a recombinant flaviviral protein of less than 100% homology to flaviviral protein to which antibodies produced in response to a subject's exposure to two or more given flaviviruses may be used.

In other embodiments, an antigen with an amino acid sequence that corresponds to only a portion of a full-length flaviviral protein, such as ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1 may be used, as may a combination of any two or more of the foregoing. For example, a truncated portion of ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1, which is recognized by antibodies generated in response to flaviviral infection, may be used. In some embodiments, an antigen may include amino acids not present in naturally occurring flaviviral proteins. For example, as would be understood by skilled artisans, for purposes of antigen synthesis and purification, an antigen may contain an amino acid tag, such as on its C-terminus or its N-terminus, without interfering with the binding thereto of an antibody produced by an individual as a result of flaviviral infection. Many possible such tags ae well-known, including a poly-histidine tag, a myc-tag, a FLAG-tag, an HA-tag, or many others, all of which could be included as part of an antigen and be within the scope of methods and kits as disclosed herein.

A sample may be a biological sample, such as a bodily fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissue homogenate, cellular extract, or spinal fluid, containing anti-flavivirus antigen antibodies, such as IgG anti-NS5, IgM anti-NS5, IgG anti-E, or IgM anti-E. A subject may be any vertebrate, such as a horse, pig, cow, dog, cat, bat, primate, including human, goat, sheep, deer, rabbit, mouse, rat, chicken or other avian species, or other animal.

EXAMPLES

The following examples are presented to further describe techniques in accordance with the method disclosed herein, but should not be read as limiting, because variations still within the scope of embodiments of the present invention will be apparent to those skilled in the art.

Reagents. Wash buffer and phosphate buffered saline pH 7.4, 0.05% sodium azide (PBS-TN) were purchased from Sigma (Sigma Aldrich, St. Louis, Mo.). Chemicals, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), were supplied by Pierce Chemicals (Pierce, Rockford, Ill.). Microspheres, calibration microspheres, and sheath fluid were obtained from Luminex Corporation (Luminex Corp., Austin, Tex.).

Serum samples. Studies were performed on serum from de-identified clinical specimens submitted to New York State Department of Health for ZIKV IgM-capture ELISA and Arbovirus MIA testing.

Positive and negative serum controls. ZIKV positive control sera were defined as positive titer from a Plaque Reduction Neutralization Test of 90% inhibition (PRNT₉₀) against ZIKV, but negative PRNT₉₀ titer against DENV. Similarly, DENV positive control sera were defined as positive PRNT₉₀ titer against DENV, but negative PRNT₉₀ titer against ZIKV. Negative control sera were defined as no ZIKV PRNT₉₀ titer as well as negative Arbovirus MIA result using WNV E protein as the diagnostic antigen (Wong et al., 2003). PBN (consisting of PBS, 1% BSA, 0.05% Sodium Azide, pH 7.4) was used as a blank control.

Expression and purification of recombinant ZIKA NS5 protein. The cDNA fragment encoding the full-length NS5 of ZIKV was amplified from an infectious clone pFLZIKV (Shan et al., 2016b), fused with a C-terminal (His)6-tag, and cloned into vector pNIC28-Bsa4 (GenBank accession EF198106), resulting in plasmid construct pNIC28-ZIKA-NS5. ZIKA NS5 protein was expressed in E. coli Rosetta 2 pLysS E. coli (Stratagene) and purified using a method as previously described (Zhao et al., 2015) with some modifications. Briefly, transformed E. coli cells was induced by 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the cell density reached OD600 of 0.6˜0.8. After incubation at 18° C. for 16 h, the cells were harvested, re-suspended in buffer A (20 mM Tris-HCl, pH 8.5, 550 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 10 mM imidazole, and 0.5×EDTA-free protease inhibitor cocktail) by sonication. The lysate was clarified by centrifugation at 40,000 g for 30 min at 4° C. The resulting supernatant was loaded onto a HisTrap Fast Flow column (GE Healthcare). The protein was eluted using a linear gradient of imidazole concentration from 40 to 500 mM. The fractions containing ZIKA NS5-(His)6 protein were pooled, concentrated, and further purified by gel filtration using a HiLoad Superdex 200 16/60 column (GE Healthcare) in buffer B (20 mM Na-Hepes, pH 8.2, 500 mM NaCl, 10% glycerol, and 5 mM DTT). The peak fractions containing ZIKA NS5-(His)6 protein were pooled and concentrated to approximately 1-2 mg/ml before storage at −80° C.

Recombinant ZIKV E, NS1, and DENV NS1 proteins. Recombinant ZIKV E, NS1, and DENV-1 to DENV-4 NS1 proteins were purchased from Meridian (Meridian Life Science, Inc., Memphis, Tenn.). All Meridian recombinant proteins were produced in insect cells and purified by affinity chromatography method. Purified proteins were analyzed by 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and stored in PBS pH 7.4.

Conjugation of protein antigens to microsphere Luminex beads. Recombinant proteins were covalently coupled to Luminex MicroPlex Microspheres carboxylated polystyrene microparticles following a previously reported protocol (Wong et al., 2003). Briefly, 50 μg of purified protein was used to couple to the surface of 6.25×106 microspheres in a two-step carbodiimide process. (i) Activation of microspheres. Microspheres were activated with 10 μl of N-hydroxysuccinimide (sulfo-NHS) (50 mg/ml) followed by 10 μl of 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide-HCl (50 mg/ml). Microspheres were then incubated for 20 min at room temperature with gentle vortexing at 10-min intervals. (ii) Coupling of recombinant proteins. Each recombinant protein was added to the activated microspheres with distinct fluorescence. Protein-microsphere mixtures were incubated for 3 h in the dark on a LabTech tube rotator (Barstead/Thermolyne, Dubuque, Iowa). The microspheres were then washed twice by centrifugation and resuspended in 1.0 ml PBS-TN [phosphate buffered saline pH 7.4, 0.05% sodium azide, 1% bovine serum albumin (BSA)]. The protein coupled microsphere were then stored at 4° C.

Multiplex reagent preparation and microsphere immunofluorescence assay (MIA) procedure. All reagent dilutions and assays were carried out in PBS-TN (phosphate buffered saline pH 7.4, 0.05% sodium azide, 1% BSA) (Sigma Aldrich, St. Louis, Mo.). The bead mixture consisted of seven beads, each coupled with a different flavivirus recombinant protein. Three bead sets contained ZIKV E (Meridian), NS1 (Meridian), and NS5 (described above). Four other bead sets contained NS1 proteins from DENV-1 to -4. The bead sets were stored at 4° C. in the dark and diluted 1:100 directly in PBS-TN before use. Biotin conjugated goat anti-human IgG/A/M affinity purified secondary antibody (Life Technologies, Grand Island, N.Y.) was diluted 1:8000 in PBS-TN directly before use. Streptavidin-R-phycoerythrin (1mg/ml SA-PE, Life Technologies, Grand Island, N.Y.) was diluted 1:100 in PBS-TN before use. All serum samples were stored at −80° C. Samples were thawed and diluted 1:100 in PBS-TN right before use. Diluted samples were used within 1 h post dilution.

A 96-well MultiScreenHTS BV 1.2 μm Filter Plate (Millipore Billerica, Mass.) was wetted with 100 μl PBS-TN and washed once with washing buffer (PBS, 0.05% Tween 20, pH 7.4; Sigma Aldrich, St. Louis, Mo.). Samples (50 μl) were dispensed in each well to which 50 μl bead mixture was added. The plate was incubated in the dark on a shaker for 30 min and washed three times with 190 μl washing buffer. After addition of 50 μl conjugate antibody, samples were incubated in the dark on a shaker for 30 min and washed three times with 190 μl wash buffer. After adding detection reagent SA-PE (50 μl), samples were incubated in the dark on a shaker for 30 min, washed twice with 190 μl wash buffer, and transferred to a flat bottom 96-well plate (Corning Incorporated, Kennebunk, Me.). Analysis was performed using a Luminex 100 Analyzer configured to count 100 beads per bead class and a 100 μl sample size.

For avidity assays, urea at a final concentration of 8 M was added to a sample during incubation of the sample with microspheres bound to a given antigen and urea incubation was for 10 min. Another microsphere-sample was incubated in the absence of urea in parallel. Median fluorescence intensity of each sample was then determined. An Avidity Index was identified, which is a ratio of median fluorescence intensity of urea-treated sample divided by median fluorescence intensity of non-urea treated sample, times 100. An IgG Avidity Index of less than or equal to 30% indicates infection occurred within the past 65 days, or early acute infection. An IgG Avidity Index of from 31% to 60% indicates infection occurred between 65 and 190 days ago, or late acute infection. An avidity index of greater than or equal to 60% indicates infection occurred more than 190 days ago. In samples in which anti-Zika NS1 or anti-Zika NS5 antibodies are detected in addition to anti-DENV antigen antibodies, avidity testing assists in determining whether one infection preceded the other, or whether one or the other infection is current or more acute than the other. For example, a subject may have a high Avidity Index for anti-DENV antigens and a low Avidity Index for anti-ZIKV antigens, indicating recent or current ZIKV infection and past DENV infection.

Results

Rationale of assay platform and antigen selection. The MIA platform for assay development was used because of (i) its multiplex capability to simultaneously detect antibodies against a number of viral proteins, (ii) rapid assay turnaround time in <4 h, and (iii) low specimen volume requirement (10 μl serum). This is in contrast with the well-established IgM-capture ELISA platform that requires >2 days of the assay turnaround time. For antigen selection, three recombinant ZIKV proteins were employed for the multiplex assay: E, NS1, and NS5. ZIKV E protein was included to sensitively detect viral infection; however, due to the cross-reactive nature of E antibodies among flaviviruses, an E-positive signal does not confirm ZIKV infection. ZIKV NS1 and NS5 proteins were included to improve assay specificity through detection of virus-type specific antibodies. In addition, recombinant DENV NS1 proteins from each of the four serotypes were included for testing virus-type specificity. All antigens, except ZIKV NS5, were commercially purchased. ZIKV NS5 was cloned, expressed, and purified to >95% homogeneity. See FIG. 1. SDS-PAGE analysis of recombinant ZIKV NS5 protein. Full-length NS5 of ZIKV was expressed in an E. coli system, and purified through affinity followed by size-exclusion chromatography (see details in Materials and Methods). The recombinant protein was analyzed on a 12% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-rad).

Establishment of multiplex MIA. Seven recombinant antigens described above (ZIKV E, NS1, NS5, and DENV-1 to -4 NS1) were individually conjugated to microsphere beads, each with a distinct fluorescent signature. A mixture of seven antigen-conjugated beads were reacted with patient serum and quantified by anti-human immunoglobulins (reactive with IgG, IgM, and IgA) with a red fluorescent phycoerythrin. To establish the cutoff level for each antigen, we assayed 20 presumed human sera from healthy individuals in the context of multiplex MIA. The results revealed cutoff values (defined as mean plus three times standard deviations) to be 1363, 284, 1905, 746, 549, 339, and 655 for ZIKV E, NS1, NS5, and DENV-1, -2, -3, and -4 NS1, respectively. These cutoff values were used to determine positive (>cutoff) and negative (<cutoff) when diagnosing patient specimens.

Stratification of patient sera. A well-defined set of patient specimens may be used to develop and verify the multiplex assay. A total of 154 patient sera with known ZIKV and DENV PRNT results were selected for assay development. Based on the PRNT results, patient sera were categorized into four distinct groups. Group I specimens (7 patients; Table 1) were both ZIKV- and DENV-negative, as defined by PRNTs <10 for both viruses.

TABLE 1 Type I specimens with neither ZIKV nor DENV infection defined by PRNT* ZIKV IgM- ZIKV DENV capture ZIKV ZIKV DENV-1 DENV-2 DENV-3 DENV-4 PRNT PRNT ELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 2 & 3 1363 284 1905 746 549 339 655 <10 <10 0.2 1189 167 573 1200 935 100 450 <10 <10 0.5 1415 155 308 220 259 61 96 <10 <10 0.9 195 54 1128 87 51 230 175 <10 <10 1.9 366 91 1900 306 216 168 136 <10 <10 1.9 7742 4619 4267 127 105 113 275 <10 <10 3.8 178 69 534 235 141 113 211 <10 <10 11.6 1001 53 211 418 509 75 125 *The table is sorted with increasing value of IgM-capture ELISA result.

Group II specimens (9 patients; Table 2) were ZIKV-negative and DENV-positive, as defined by ZIKV PRNT<10 and DENV PRNT>10.

TABLE 2 Type II specimens with DENV only infection defined by PRNT* ZIKV IgM- ZIKV DENV capture ZIKA ZIKV DENV-1 DENV-2 DENV-3 DENV-4 PRNT PRNT ELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 2 & 3 1363 284 1905 746 549 339 655 <10 20 0.19 9131 234 170 5375 1579 3091 1442 <10 40 0.60 7145 211 331 9101 2831 5415 3740 <10 80 1.5 11139 2514 249 9145 6658 10609 14941 <10 160 1.5 9596 1806 177 7486 5198 8241 11867 <10 640 1.5 5381 46 227 5323 860 2235 1352 <10 80 1.6 8071 67 554 6862 510 1765 658 <10 40 5.1 6017 51 1473 98 50 99 165 <10 160 5.8 7756 38 968 4259 179 582 305 <10 40 77 8703 106 1009 8805 1511 3165 1558 *The table is sorted with increasing value of IgM-capture ELISA result.

Group III specimens (43 patients; Table 3) were ZIKV-positive and DENV-negative, as defined by ZIKV PRNT>10 and DENV PRNT <10.

TABLE 3 Type III specimens with ZIKV only infection defined by PRNT * ZIKV IgM- ZIKV DENV capture ZIKA ZIKV DENV-1 DENV-2 DENV-3 DENV-4 PRNT PRNT ELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 2 & 3 1363 284 1905 746 549 339 655 80 <10 0.6 9960 5207 2624 1346 1172 454 744 320 <10 0.99 10692 6173 1676 409 531 660 631 2560 <10 1.8 16318 9407 10423 548 737 802 492 320 <10 1.9 12377 6550 21510 391 644 927 422 40 <10 2.4 858 617 1401 349 272 249 374 10 <10 2.75 2185 297 1777 795 729 189 285 80 <10 3.04 800 481 227 163 213 118 252 160 <10 3.80 9088 7010 3298 438 588 782 459 320 <10 3.97 11272 4849 2129 1556 1268 250 838 320 <10 4 11358 4933 2179 1503 1212 212 735 320 <10 4.5 8592 5500 4362 311 455 452 220 2560 <10 4.5 8592 5500 4362 311 455 452 220 160 <10 6.2 3896 3152 2524 574 477 203 371 160 <10 7.88 5782 701 574 340 260 125 217 1280 <10 7.9 3233 5196 5865 231 263 315 246 1280 <10 7.9 4777 6771 8260 364 417 508 381 320 <10 8.1 8842 4811 15599 214 349 561 276 320 <10 8.1 9768 5001 18463 253 405 532 294 160 <10 8.29 457 540 930 464 258 204 168 1280 <10 10.9 496 1010 3139 102 84 74 108 160 <10 13.5 12420 7611 1809 141 195 218 401 160 <10 13.5 14292 8757 2491 180 243 265 548 160 <10 13.7 7432 6664 2876 90 159 337 149 40 <10 17 536 1554 2612 153 202 122 153 640 <10 17.70 6381 7713 18520 1095 1082 628 419 640 <10 20.1 2474 2312 2038 197 205 67 82 640 <10 20.18 3703 1566 4269 75 69 314 54 160 <10 22 530 428 679 151 116 110 119 160 <10 22 999 5442 3802 217 234 263 86 320 <10 25 7905 6814 5828 244 350 242 166 320 <10 25.5 8518 6914 6650 261 378 303 228 1280 <10 27 8494 5334 3631 102 107 103 274 1280 <10 27.18 10443 6998 4530 157 163 158 407 640 <10 28 3488 11213 4269 290 452 579 62 640 <10 28.8 8741 7471 3820 278 492 662 147 640 <10 29.9 6212 4915 5969 193 221 288 115 640 <10 30 3998 3187 3867 133 141 158 73 1280 <10 32 11745 7674 4459 174 49 242 250 1280 <10 32.3 13259 8154 4726 227 309 304 309 640 <10 42.1 9721 7456 658 143 276 230 139 2560 <10 47.2 16346 4928 3714 149 158 320 60 320 <10 50.4 8250 7160 638 134 251 215 122 320 <10 65.6 3645 2785 1229 52 74 77 79 * The table is sorted with increasing value of IgM-capture ELISA result.

Group IV specimens (95 patients; Table 4) were both ZIKV- and DENV-positive, as defined by PRNTs>10 for both viruses.

TABLE 4 Type IV specimens with ZIKV and/or DENV infection(s) defined by PRNT* ZIKV IgM- ZIKV DENV capture ZIKV ZIKV DENV-1 DENV-2 DENV-3 DENV-4 PRNT PRNT ELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 2 & 3 1363 284 1905 746 549 339 655 2560 320 0.3 12411 3002 6118 7992 5677 11340 10357 10 40 0.40 7446 285 790 7714 2875 5725 4420 320 2560 0.4 15916 4164 11951 10070 8345 14047 13683 20 1280 0.41 18428 802 461 7969 5627 10631 9581 80 640 0.45 17887 2203 665 5073 3700 5778 9545 160 640 0.45 18357 2550 796 5886 4181 6677 10700 20 640 0.45 17361 874 551 9206 6339 11251 11171 10 80 0.49 16355 1950 570 10920 8275 15354 11740 1280 2560 0.53 18809 6682 650 16472 10777 18272 19435 10 160 0.53 8131 844 325 5142 3838 7463 6020 1280 2560 0.56 20863 6548 602 16500 10369 19309 19308 160 2560 0.62 17472 3453 2503 12655 2806 11109 16787 160 2560 0.69 18220 3461 2384 13241 2871 12265 17857 2560 640 0.8 18354 8434 2396 382 426 556 309 640 1280 1.09 16382 4065 3303 11394 7116 13885 7695 320 2560 1.3 17322 4085 875 15507 11175 17637 16700 2560 320 1.41 19995 6926 8456 12524 11268 17951 16740 640 1280 1.53 21501 7952 3037 16148 12733 19792 22445 320 160 1.63 14009 6104 1374 11801 11004 17719 4475 320 2560 1.7 19788 8615 3494 16160 13589 19342 20432 160 2560 1.8 16928 3607 551 14462 10561 17158 15002 1280 1280 1.81 17896 7868 2698 14294 11672 17853 21180 320 2560 1.9 20374 8309 2755 16466 13975 19997 20689 640 2560 2.1 21021 12154 15783 18993 14796 21073 22419 640 2560 2.16 21035 11444 13536 18240 15675 21260 22412 5160 5120 2.3 18554 6707 553 12751 10464 17362 20153 5120 1280 2.3 19606 11677 11105 14953 11886 18231 21051 5120 1280 2.32 18697 9335 7705 12257 9981 16633 17776 640 1280 2.45 20559 10939 21381 14675 10696 18537 17415 2560 1280 2.5 19945 9641 13280 15589 13022 19484 15561 2560 1280 2.52 18959 8035 11647 14771 12827 19260 13601 320 40 2.57 9760 1309 386 5967 1610 2724 3719 160 40 2.59 10893 859 180 3100 914 1643 6687 160 40 2.72 13602 1082 205 4179 1260 2191 8609 320 40 2.99 10133 1442 417 6469 1696 3018 4257 320 5120 3.13 18591 7977 1050 11995 9617 15868 14587 160 320 3.53 19680 5587 1681 317 292 543 270 2560 2560 3.89 17268 4501 5464 13748 9869 17527 12169 5120 5120 3.99 21644 10859 21825 12761 9859 17755 20283 160 320 4.02 16822 6103 2070 14021 10298 17324 15517 640 1280 4.42 20593 5338 1739 12752 11158 18128 18042 1280 1280 4.43 19973 8594 11143 12763 11051 17739 16997 1280 320 4.79 19355 10636 9796 12751 10778 18003 18073 640 1280 4.88 16101 4858 776 13093 8198 14431 8430 2560 1280 5.01 20780 12080 17177 16920 13760 20359 17012 160 640 5.1 19275 6311 2744 14623 12984 19646 15016 160 640 5.34 16289 5747 2635 15009 11904 18826 16649 10 80 5.47 16794 1307 1328 389 200 346 281 1280 640 6.05 16809 6987 3429 13666 12416 18579 5716 1280 80 6.52 2111 3626 1100 209 289 243 291 320 160 7.04 19585 2905 3390 10849 5579 10368 11556 320 160 7.04 20334 3419 4461 11917 6275 12136 12769 320 80 7.32 19940 2687 2942 10583 5368 9833 10471 1280 10 7.49 13338 9654 6618 462 650 1220 700 1280 10 7.5 12624 9195 6717 341 478 986 514 2560 20480 8.3 20265 9171 2720 16781 12747 18712 20473 640 5120 8.5 20671 9098 7078 17100 14388 18608 22061 640 10240 9.04 21114 11671 11204 19358 15223 21543 22106 2560 320 10.16 18279 9899 7213 4524 3165 6948 1278 160 640 10.90 20183 7660 2078 2723 2628 5909 1384 320 640 10.99 14737 4336 15626 11828 9458 15927 6841 320 2560 11.1 20977 10041 2254 17295 13477 19549 10638 640 10240 11.4 20785 12169 11619 18884 15119 20667 21989 1280 2560 12 19968 8988 4259 17044 13238 19273 22230 1280 2560 12.54 20467 8901 3673 16470 13221 19341 22846 320 640 13 20546 6045 1818 15929 10974 18017 3192 640 1280 14 18531 5202 1896 11359 10329 16770 16440 640 640 14.64 20336 7675 1722 13716 12002 18849 20055 320 2560 15 21156 10463 3187 18463 14572 20246 22876 640 2560 16 20527 9862 5041 16141 12408 19080 21991 640 2560 16.29 19340 9066 5185 15029 11748 17407 20104 2560 20480 17 22317 6665 7447 15855 10725 17403 5509 2560 20480 17.23 17999 5738 7214 13635 8930 14737 5748 160 1280 17.41 19848 8375 6839 15679 13745 18998 22484 160 160 20.73 9391 6074 3340 356 679 566 309 10240 320 22 17752 7270 4264 62 156 388 96 1280 1280 22.2 20185 8584 16360 16030 12594 20100 10783 1280 20480 23 20703 10096 7859 18086 13614 18507 21267 320 20 23.6 9924 8081 9837 113 203 469 231 1280 5120 23.69 18836 8498 4119 15799 12910 18325 20333 320 10 25 1061 3108 2294 88 89 83 894 640 640 25.23 20066 9245 4529 17030 14304 20372 22431 2560 2560 26.13 21184 9524 2734 15615 13845 19053 22401 640 2560 27.9 19121 3787 3230 3522 3308 7426 3591 1280 10240 29.15 19711 10327 2437 16973 14262 18898 20341 1280 2560 29.9 21902 8590 3728 16006 13452 20836 22510 640 1280 30.53 18080 8675 17545 16414 13525 18677 14452 2560 640 32 20207 6094 11566 10231 7930 14480 11206 640 2560 33.4 20649 10126 6628 16806 13194 18759 22293 320 160 35.88 4640 4268 2211 294 263 175 169 320 10 44.8 9837 7200 2590 284 253 308 194 2560 2560 46.1 19943 7114 7170 15766 11664 18454 8917 640 640 47.93 2828 5437 7229 334 1185 1407 299 1280 20 49.11 14063 6262 6839 359 291 193 250 1280 640 57 19062 6018 11756 12258 10914 17240 17024 *The table is sorted with increasing value of IgM-capture ELISA result.

It should be noted that, due to cross-neutralization of antibodies among flaviviruses, group IV specimens could derive from patients who were (i) infected with both ZIKV and DENV, (ii) infected with ZIKV only but had antibodies cross-reactive to DENV, or (iii) infected with DENV only but with antibodies cross-reactive to ZIKV.

Multiplex MIA and IgM-capture ELISA diagnosis. Patient samples were subjected to multiplex MIA and the well-established IgM-capture ELISA (Martin et al., 2000). Tables S1 to S4 summarize the results for groups I to IV specimens, respectively. Each specimen is presented with results from PRNT, IgM-capture ELISA, and multiplex MIA for individual antigens. It should be pointed out that, for IgM-capture ELISA, P/N<2 is defined as negative, P/N 2-3 as equivocal, and P/N>3 as positive. Table 5 in FIG. 2 summarizes the overall diagnostic results. For Table 5 in FIG. 2, results from Tables 1 to 4 are summarized for comparison of PRNT, IgM-capture ELISA, and multiplex MIA diagnosis. For each diagnostic parameter, the total number of samples that were diagnosed as “positive” (greater than cutoff line) or “negative” (less than cutoff line) is indicated, followed by its corresponding percentage of the total number of specimens from that specific specimen group. Percentage (%)=(number of positive or negative specimens/total number of specimen from the specific specimen group)×100%.

For group I specimens (neither ZIKV nor DENV infection), both ZIKV IgM-capture ELISA and E MIA showed 71% negative; the MIA results from ZIKV NS1, ZIKV NS5, and combined DENV-1 to -4 NS1 showed 86% negative. For group II specimens (DENV infection only), ZIKV IgM-capture ELISA showed 67% negative (i.e., 33% cross-reactivity with DENV); ZIKV E and combined DENV-1 to -4 NS1 MIA showed 100% and 89% positive, respectively; in contrast, ZIKV NS1 and NS5 MIA showed 78% and 100% negative, respectively. For group III specimens (ZIKV infection only), ZIKV IgM-capture ELISA showed 86% positive; ZIKV E, NS1, and NS5 MIA showed 84%, 100%, and 74% positive, respectively; whereas combined DENV-1 to -4 NS1 MIA showed 63% negative (i.e., 37% cross-reactivity with ZIKV). For group IV specimens (at least one infection from ZIKV and/or DENY), ZIKV IgM-capture ELISA, E, NS1, NS5, and combined DENV NS1 showed 63%, 99%, 100%, 73%, and 96% positive, respectively. These results enable the following analysis.

(I) Comparison of ZIKV IgM-capture ELISA and E MIA. Compared with IgM-capture ELISA, ZIKV E MIA alone showed equivalent accuracy when diagnosing group III specimens, with 86% and 84% of the samples tested positive from IgM-capture ELISA and E MIA, respectively. When diagnosing group IV specimens, the E MIA showed better sensitivity than IgM-capture ELISA, with 63% and 99% of specimens tested positive, respectively. Two factors may account for this improvement. (i) MIA measures IgG and IgA in addition to IgM, whereas IgM-capture ELISA does not capture IgG and IgA. (ii) The amount of IgM declines after the convalescent phase of ZIKV infection; therefore, specimens (taken long after convalescent phase) may have low levels of IgM and high levels of IgG, which is not detected by the IgM-capture ELISA. Taken together, the results indicate that E MIA alone has equivalent or better sensitivity than IgM-capture ELISA.

(II) Relative specificity of ZIKV E, NS1, and NS5 MIA. Comparison of the results from ZIKV E, NS1, and NS5 MIA demonstrates that antibody response to NS1 and NS5 antigens is more ZIKV-specific than that to E antigen. Specifically, ZIKV E MIA showed 100% cross-reactivity with specimens with DENV only infection from group II, confirming the cross-reactive nature of flavivirus E antibodies. In contrast, ZIKV NS1 MIA showed 14% and 22% false positive results when testing groups I and II specimens, but 100% positive accuracy when analyzing groups III and IV specimens. For ZIKV NS5 MIA, the assay exhibited 14% and 0% false positive results when testing groups I and II specimens, and 74% and 73% positive accuracy when analyzing groups III and IV specimens. The results clearly indicate that inclusion of ZIKV NS1 and NS5 in the MIA could improve the diagnostic accuracy when compared with the MIA that uses E protein alone.

(III) Cross reactivity between DENV/ZIKV NS1 proteins and their antibodies. Although antibody response to ZIKV NS1 is more virus-type specific than that to E protein (see above), we clearly observed cross reactivity between DENV and ZIKV NS1 proteins and their antibodies. Specifically, DENV NS1 MIA showed 89% and 96% positive accuracy when testing groups II and IV specimens, respectively; and 14% and 37% false positive results when testing groups I and III specimens, respectively. The 37% false positive result demonstrates that DENV NS1 cross-reacts to specimens with ZIKV-only-infection. Reciprocally, ZIKV NS1 MIA exhibited 22% false positive when testing specimens with DENV-only-infection from group II. Altogether, the results showed 22-37% cross reactivity between DENV and ZIKV NS1 proteins. The data are in agreement with the recent report that antibodies to NS1 are largely ZIKV-specific.

Conventionally, ZIKV serologic diagnosis is mainly based on IgM-capture ELISA with Emergency Use Authorization (EUA) approval from FDA. There is a need for improved methods for detection of anti-ZIKV antibodies in samples from a subject. As disclosed herein, the diagnostic power of viral envelope protein (that elicits robust, yet cross-reactive antibodies to other flaviviruses) is hereby combined with the differential power of viral nonstructural proteins NS1 and NS5 (that induce more virus-type specific antibodies). Disclosed herein is use of an assay using an MIA format that can shorten assay turnaround time. Over 10,000 sera have been tested by conventional, presently FDA emergency use authorized Zika MAC-ELISA testing. Any such samples presumptive as positive for Zika IgM according to such assay was further tested via PRNT. Approximately 80% of the time such assays resulted in identifying the sample as indicating “undifferentiated flavivirus” (meaning indicating that some flaviviral infection had been present, but not definitively ruling in or out ZIKV or DENV. Further testing on over 3600 of these samples permitted an identification of whether a subject had been infected with ZIKV, DENV, both, or neither, and whether in the past 65 days, past 65-190 days, or more than 190 days ago. As a result, as opposed to high rates of “undifferentiated flavivirus” results, the method disclosed herein permitted confirmatory identification of such specifics of infection in approximately 90% of cases where flaviviral infection was indicated.

Furthermore, an assay disclosed herein was performed on many Zika IgM nonreactive, PCR negative specimens from pregnant women with known dengue history. Nearly half of 199 samples from such pregnant women had evidence of a history of Zika infection at some time according to an assay disclosed herein, combining detection of anti ZIKV E, anti-ZIKV NS1, and anti-DENV1-4 NS1 antibodies. Some pregnant women who are IgM negative to Zika may still deliver an infant at risk of congenital Zika syndrome, indicating the importance of accurate testing such as disclosed herein.

Using over 3600 samples patient samples with known ZIKV and DENV PRNT results, the method was verified and improved ability to distinguish ZIKV infection from DENV infection according to methods and kits as disclosed herein. Embodiments of a method disclosed herein are distinct from the single antigen-based (either E or NS1) diagnostic assays, including the E-based IgM-captured ELISA from InBios (with EUA approval from FDA), NS1-based indirect ELISA from EuroImmun (approved for clinical use in Europe), and NS1-based IgM-capture ELISA from NovaTec (currently for investigational research use).

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be understood that the terms “comprise”, “have”, “include”, and “contain” (and any related variants thereof) are open-ended linking verbs. As a result, a method, step, or device that “comprises”, “has”, “includes” or “contains” one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more steps or elements. The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below, if any, are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The disclosure herein is illustrative and not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Embodiments are described to best explain the principles of one or more aspects of the invention and the practical application, and to enable others of ordinary skill in the art to understand one or more aspects of the invention for various embodiments with various modifications as are suited to the particular use contemplated. Further details pertaining to the present disclosure can be found in Appendix A, attached hereto and incorporated herein in its entirety.

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1-32. (canceled)
 33. A method, comprising: contacting a first portion of a sample from a subject with a first suspension comprising a first plurality of microspheres, wherein the sample comprises blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissue homogenate, cellular extract, or spinal fluid, and individual microspheres are conjugated to Zika virus (ZIKV) peptides that differ from ZIKV peptides to which other individual microspheres are conjugated, and the ZIKV peptides are selected from the group consisting of full-length ZIKV NS1, full-length ZIKV NS5, and full-length ZIKV envelope protein; forming a first incubated suspension wherein forming comprises incubating said first portion with said first suspension, in the presence of between 6 M urea and 10 M urea, to permit high-avidity binding between anti-ZIKV NS1, anti-ZIKV NS5, and anti-ZIKV envelope protein antibodies present in the first portion and microspheres of said first plurality of microspheres; forming a second incubated suspension wherein forming comprises contacting said first plurality of microspheres with an anti-ZIKV antibody detecting-reagent to permit binding between the anti-ZIKV antibody detecting reagent and antibodies bound to microspheres of said first plurality of microspheres; removing from the second incubated suspension anti-ZIKV antibody detecting-reagent molecules that are not bound to antibodies bound to microspheres; and detecting anti-ZIKV antibody detecting-reagent molecules in the second incubated suspension.
 34. The method of claim 33, wherein the ZIKV peptides are full-length ZIKV NS1 and full-length ZIKV NS5.
 35. The method of claim 33, wherein individual microspheres are conjugated to full-length ZIKV NS1.
 36. The method of claim 33, wherein individual microspheres are conjugated to full-length ZIKV NS5.
 37. The method of claim 33, wherein the ZIKV peptides are full-length ZIKV NS1 and full-length ZIKV envelope protein.
 38. The method of claim 33, wherein the ZIKV peptides are full-length ZIKV NS1, full-length ZIKV NS5, and full-length ZIKV envelope protein.
 39. The method of claim 33, wherein detecting comprises measuring an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent, an autofluorescence signal emitted by a microsphere, or an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent and an autofluorescence signal emitted by a microsphere.
 40. The method of claim 37, further comprising detecting the presence of an antibody to a Dengue virus (DENV) protein in a sample, wherein the protein comprises NS1 and the DENV comprises DENV1, DENV2, DENV3, DENV4, or any combination of two or more of the foregoing.
 41. The method of claim 38, further comprising detecting the presence of an antibody to a Dengue virus (DENV) protein in a sample, wherein the protein comprises NS1 and the DENV comprises DENV1, DENV2, DENV3, DENV4, or any combination of two or more of the foregoing.
 42. The method of claim 33, wherein said first portion is incubated with said first suspension in the presence of between 7 M urea and 9 M urea.
 43. The method of claim 33, further comprising: contacting a second portion of the sample with a second suspension comprising a second plurality of microspheres, wherein the sample comprises blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissue homogenate, cellular extract, or spinal fluid, and individual microspheres are conjugated to Zika virus (ZIKV) peptides that differ from ZIKV peptides to which other individual microspheres are conjugated, and the ZIKV peptides are selected from the group consisting of full-length ZIKV NS1, full-length ZIKV NS5, and full-length ZIKV envelope protein; forming a third incubated suspension wherein forming comprises incubating said second portion with said second suspension, in the absence of urea, to permit binding between anti-ZIKV NS1, anti-ZIKV NS5, and anti-ZIKV envelope protein antibodies present in the second portion to and microspheres of said second plurality of microspheres; forming a fourth incubated suspension wherein forming comprises contacting said second plurality of microspheres with an anti-ZIKV antibody detecting-reagent to permit binding between the anti-ZIKV antibody detecting reagent and antibodies bound to microspheres of said second plurality of microspheres; removing from the fourth incubated suspension anti-ZIKV antibody detecting-reagent molecules that are not bound to antibodies bound to microspheres; detecting anti-ZIKV antibody detecting-reagent molecules in the fourth incubated suspension; and determining an avidity index, wherein determining comprises comparing an amount of anti-ZIKV antibody detecting-reagent molecules detected in the second incubated suspension with an amount of anti-ZIKV antibody detecting-reagent molecules detected in the fourth incubated suspension
 44. The method of claim 43, wherein the ZIKV peptides are full-length ZIKV NS1 and full-length ZIKV NS5.
 45. The method of claim 43, wherein individual microspheres are conjugated to full-length ZIKV NS1.
 46. The method of claim 43, wherein individual microspheres are conjugated to full-length ZIKV NS5.
 47. The method of claim 43, wherein the ZIKV peptides are full-length ZIKV NS1 and full-length ZIKV envelope protein.
 48. The method of claim 43, wherein the ZIKV peptides are full-length ZIKV NS1, full-length ZIKV NS5, and full-length ZIKV envelope protein.
 49. The method of claim 43, wherein detecting comprises measuring an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent, an autofluorescence signal emitted by a microsphere, or an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent and an autofluorescence signal emitted by a microsphere.
 50. The method of claim 47, further comprising detecting the presence of an antibody to a Dengue virus (DENV) protein in a sample, wherein the protein comprises NS1 and the DENV comprises DENV1, DENV2, DENV3, DENV4, or any combination of two or more of the foregoing.
 51. The method of claim 48, further comprising detecting the presence of an antibody to a Dengue virus (DENV) protein in a sample, wherein the protein comprises NS1 and the DENV comprises DENV1, DENV2, DENV3, DENV4, or any combination of two or more of the foregoing.
 52. The method of claim 43, wherein said first portion is incubated with said first suspension in the presence of between 7 M urea and 9 M urea. 